甘蓝型油菜BcNA1基因启动子在转基因烟草中对GUS基因表达的调控

通过PCR扩增,从甘蓝型油菜（Brassica     

Brassica S-Proteins Accumulate in the Intercellular Matrix along the Path of Pollen Tubes in Transgenic Tobacco Pistils

A tobacco plant transformed with a Brassica oleracea SLG-22 gene was analyzed by immunocytochemical methods to determine the localization of the transgene-encoded protein product. Immunolabeling was observed in the pistil along the path followed by pollen tubes after pollination. S-antigen accumulated in the intercellular matrix of the transmitting tissue of the style and its continuation in the basal portion of the stigma and outside a few special cells of the placental epidermis of the ovary. This pattern of S-antigen distribution closely resembles that described for the S-associated glycoproteins of self-incompatible Nicotiana alata and differs from its distribution in B. oleracea.     

In situ Localization of Calmodulin mRNA and Protein in the Developing Anthers and Pistils in Rice

The temporal and spatial distribution patterns of calmodulin mRNA and protein were detected by in situ RNA hybridization and in situ immunohistochemical localization, respectively, in the developing anthers and pistils in rice ( Oryza sativa L. cv. Chunjiang). Calmodulin (CAM) gene was substantially expressed in the tapetum, stigma, pollen tube track, degenerated synergid and transfusion parenchyma cells. Less but significant amounts of CAM were also localized in the microspore mother cells, microspores, pollen, antipodal cells, egg cell and central cell. The density of reaction products varied with different developmental stages. During the earlier developmental stages of anther, CAM gene was expressed strongly, then declined gradually and became centralized in some special sites such as the tapetmn, pollen germination apertures, etc. During the embryogenesis, CAM gene was expressed stronger in the endosperm cells than in the proembryo cells at the earlier stage but it was reversed at the stage of embryo differentiation. The authors propose that CAM may be involved in regulating such events as microspore development, pollen germination, pollen tube growth, fertilization, and substance transport during sexual plant reproduction through Ca2 +-CAM signaling pathways.     

Transgenic Brassica campestris Plants Expressing the gfp Gene

A protocol has been worked out for efficient regeneration and genetic transformation of summer rape (Brassica campestris L. var. oleifera). Cotyledons of the 5-day-old seedlings were transformed with Agrobacterium tumifaciens strain AGL cells comprising a binary vector with a selectable neomycin phosphotransferase II gene and a reporter gene encoding green fluorescent protein (GFP). Explants were cultured on a regeneration MS medium supplemented with ABA, and transformants were selected on the same medium (minus ABA and plus 70 mM AgNO₃ and 15 mg/l kanamycin). The frequency of shoot regeneration on explant petioles was 30–40%. Transgenic plants were identified by GFP fluorescence and by polymerase chain reaction and Western blotting analysis. The transformation efficiency was as high as 75% of the total number of regenerated shoots.     

玉米茎特异启动子克隆及其在转基因烟草中的活性分析

从玉米自交系‘综31’基因组中分离了1个茎特异表达启动子,命名为ZmSSP。用ZmSSP替换植物表达载体pCAMBIA3301的CaMV35S启动子,构建了ZmSSP驱动GUS报告基因的重组表达载体pCAMBIA3301-ZmSSP-GUS,并采用农杆菌介导法转化烟草,对转基因烟草营养器官中GUS表达模式进行了分析。结果表明:在烟草中ZmSSP活性低于CaMV35S启动子;不同转基因株系中ZmSSP活性及模式有显著差异;10个转基因株系统计结果表明,GUS表达量最高的营养器官是叶柄,平均是Actin基因表达量的2.71倍;其次是叶片和茎,在根中的GUS表达量最低,平均是Actin基因表达量的29.6%,是叶柄中活性的10.9%。研究认为,ZmSSP是较好的组织特异性启动子,适用于通过植物基因工程技术驱动目的基因进行地上营养器官的性状改良。     

嗜铬颗粒蛋白A基因的反义转基因小鼠模型的建立及该基因启动子的组织特异性

构建小鼠嗜铬颗粒蛋白A（Chromogranin,A,CGA)基因的反义DNA载体pGAS1C－lacZ。用电穿孔的方法将该载体转化大鼠肾上腺髓质细胞瘤细胞系PC－12,X－Gal染色后证明位于CGA基因启动子下游的报告基因lacX已经表达。用限制性内切酶除去载体的质粒骨架后,显微注射入供体昆明小鼠的受精卵中,随后将注射过DNA的受精卵移植入假母的输卵管中,完成正常的胚胎发育。用PCR的方法筛选假母产下的小鼠,得到CGA基因反义RNA基因首建鼠14只。将首建鼠分别与正常昆明鼠交配,产生后代。取首建鼠的肾上腺进行X－Gal染色,组织用于石蜡切片,根据各鼠肾上腺切片的蓝色深浅判定转入基因量的高低,筛选到两只表达量高的首建鼠,留下它们的后代。取转基因鼠的各种组织用于X－Gal染色,发现报告基因在肾上腺、胰腺的胰岛中有表达,而在肌肉、脂肪组织中无表达,说明CGA基因的启动子具有神经内分泌组织特异性。     

甘露碱合成酶基因启动子调控的萤光素酶基因在转化烟草中的表达

利用我们自己分离的甘露碱含成酶基因启动子与萤光素酶结构基因、胭脂碱合成酶基因’3末端结构相拼构成一融合基因,并在带有此融合基因的中间载体PBZ7610插入Ti质粒T区的tmr基因,构成中间载体pBZ7621。利用改建的Ti质粒载体PGV3850,将萤光素酶融合基因引入了烟草植株,结果表明,萤光素酶融合基因在转化烟草中能表达。中间载体pBZ7610还带有PstⅠ,HindⅢ,XbaⅠ等多个单一的酶切位点,外源基因极易插入。利用中间载体pBZ7621,还可研究启动子在高等植物不同发育阶段中的功能特征。     

小鼠超高硫角蛋白基因启动子的克隆及其表达活性分析

目的筛选在小鼠毛囊中具有高表达活性的内源启动子,为建立小鼠被毛特异表达外源蛋白的转基因技术奠定基础。方法以小鼠基因组为模板,克隆得到超高硫角蛋白基因启动子超高硫角蛋白(UHS),将其分别与pβgal-Basic和pAcGFP1-N1载体连接,构建了重组真核表达载体。采用阳离子脂质体法转染胎鼠组织块,分析其表达活性。结果转染后48 h,在蓝光激发条件下可以检测到绿色荧光蛋白(GFP)在小鼠毛囊区高表达,转染96 h后,表达减弱;此外,转染后48 h,βgal染色结果显示在皮肤块的毛囊区存在蓝色点状区域。结论 UHS启动子在小鼠毛囊中具有表达特异性。     

Expression of 2S Seed Storage Protein Gene of Brassica juncea in Transgenic Tobacco Plants under Constitutive and Seed-specific Promoters

The coding and upstream promoter regions of Brassica juncea 2S seed storage protein gene were amplified by polymerase chain reaction. The plant expression cassettes containing 2S seed storage protein gene under the control of either a constitutive Caulimovirus 35S promoter or a seed specific 2S protein promoter and the polyadenylation signal of a pea rbcS gene were used for Agrobacterium — mediated transformation of tobacco (Nicotiana tabacum cv Petit Havana). Integration of 2S gene was confirmed by Southern blot and PCR analysis of plant genomic DNA. Expression of introduced 2S protein gene was monitored by slot blot analysis of total RNA using 2S protein sequence as hybridization probe and also by immunodot blot analysis using specific antiserum of 2S protein. Expression was either constitutive with CaMV 35S promoter or highly seed-specific with Brassica 2S promoter.     

Tissue-Dependent Expression of the Rice wx+ Gene Promoter in Transgenic Rice and Petunia

The waxy (wx) locus, which controls the amylose synthesis, isknown to be expressed specifically in the endosperm and pollen.To study the tissue-specific regulation of the wx⁺ gene, weintroduced a fusion gene that consisted of the upstream sequenceof the wx⁺ gene and the gene for rß-glucuronidase(GUS) into cells of rice (Oryza sativa L.) and petunia (Petuniahybrida L.). GUS activity was examined in the regenerated transgenicrice and petunia plants. In transgenic rice, the upstream sequenceof the wx⁺ gene was sufficient to direct the tissue-specificexpression of GUS in the endosperm and pollen, and the controlof expression was quantitative. By contrast, in transgenic petunia,the same fusion gene was expressed in pollen but not in theendosperm. These results suggest that the putative cis-actingelements that direct pollen-specific expression are common toor similar in both monocotyledonous and dicotyledonous plants,whereas ciy-elements responsible for the endosperm-specificexpression of the rice wx⁺ gene do not function in petunia,in which development of the endosperm differs from that in rice. ⁴Present address: Division of Biological Sciences, GraduateSchool of Science, Hokkaido University, Kita-ku, Sapporo, 060Japan     

玉米醇溶贮藏蛋白基因启动子PCR扩增及其驱动GUS基因在转基因烟草种子中的表达

以玉米(Zea mays L.)黄化苗为材料,利用PCR技术扩增了玉米19kDa醇溶贮藏蛋白基因(zein)起始密码子上游启动子片段,序列分析结果表明,克隆的-1～-694片段具有19kDa zein启动子特点,与同一家族中其它基因的对应区段同源性达90％以上。将此启动子插入pPKGT的GUS基因及NOS终止子上游构成表达载体。经农杆菌转化烟草(Nicotiana tabaccum Var.samsum),得到了转化植株。转化的烟草的PCR扩增及Southern杂交证明目的片段已整合到烟草基因组中。转基因植株的GUS活性检测表明,在叶、根中无GUS活性,GUS活性只存在于种子中。转基因植株烟草种子经冷冻切片,GUS底物Xgluc活体组织染色证明GUS活性只存在一层介于种子胚乳与种皮之间的细胞中。     

Resistance to BmNPV via Overexpression of an Exogenous Gene Controlled by an Inducible Promoter and Enhancer in Transgenic Silkworm, Bombyx mori

The hycu-ep32 gene of Hyphantria cunea NPV can inhibit Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication in co-infected cells, but it is not known whether the overexpression of the hycu-ep32 gene has an antiviral effect in the silkworm, Bombyx mori. Thus, we constructed four transgenic vectors, which were under the control of the 39 K promoter of BmNPV (39 KP), Bombyx mori A4 promoter (A4P), hr3 enhancer of BmNPV combined with 39 KP, and hr3 combined with A4P. Transgenic lines were created via embryo microinjection using practical diapause silkworm. qPCR revealed that the expression level of hycu-ep32 could be induced effectively after BmNPV infection in transgenic lines where hycu-ep32 was controlled by hr3 combined with 39 KP (i.e., HEKG). After oral inoculation of BmNPV with 3 × 10(5) occlusion bodies per third instar, the mortality with HEKG-B was approximately 30% lower compared with the non-transgenic line. The economic characteristics of the transgenic lines remained unchanged. These results suggest that overexpression of an exogenous antiviral gene controlled by an inducible promoter and enhancer is a feasible method for breeding silkworms with a high antiviral capacity.     

一种受抗生素诱导的启动子的构建及活性分析

多聚ADP核糖聚合酶（PARP）受基因毒剂的特异性诱导。将拟南芥（Arabidopsis thaliana）AtPARP1基因上游长2179bp的启动子片段插入到质粒pAKK687的β-葡萄糖醛酸糖苷酶（GUS）报告基因上游,转化拟南芥。GUS组织化学染色结果表明,GUS报告基因仅在苗龄3-5天的拟南芥根部及花发育早期的雄蕊中表达;1.5μg.mL-1博莱霉素与22μg.mL-1丝裂霉素联用强烈诱导了GUS报告基因的表达（尤其在拟南芥的幼苗和果荚中）。进一步降低抗生素浓度,发现单独使用1μg.mL-1博莱霉素对GUS报告基因也具较强的诱导活性,且对拟南芥幼苗的生长无影响。上述结果表明,AtPARP1启动子是一个新型的具较大应用潜力的抗生素诱导型启动子。     

Promoter Activity of Phenylalanine Ammonia-Lyase Gene of Pharbitis nil in Arabidopsis

Promoter activity of phenylalanine ammonia-lyase (PAL) gene of Pharbitis nil was examined by introducing a PAL:GUS construct into Arabidopsis. GUS staining was observed in vascular bundles of hypocotyl and cotyledons, endodermal cells of the primary root, hydathodes, stigma and pollens of mature flower, abscission zones of petals and sepals and inner layer of seed coat. Light induced GUS expression in cotyledons and the upper part of hypocotyl in which anthocyanin was accumulated. Wounding also induced GUS expression. Endogenous PAL activity increased earlier than the GUS activity directed by the PAL promoter.     

Runx2参与调控Osterix 启动子活性及其基因表达

尽管Runx2和Osterix都是成骨细胞分化途径中关键的转录因子,但是Runx2是否能够调控Osterix,还不为所知.研究发现,在非成骨细胞系,无论是间充质干细胞还是已分化的细胞,以及成骨细胞系中,Runx2都能诱导Osterix的表达.同时Runx2能够上调3.2kb人的Osterix基因启动子活性.进一步实验证明,在这一段启动子中存在Runx2功能性的结合位点.因而,实验结果有力地支持了这样一个假设,即Runx2参与了Osterix基因的表达调控.瞬时转染和荧光素酶双报告分析结果显示,在非成骨细胞中,Osterix明显上调2.3kb的Ⅰ型胶原蛋白启动子活性,但Runx2却不能.这样的差别暗示,在成骨细胞分化过程中位于Runx2下游的转录因子Osterix是刺激Ⅰ型胶原蛋白基因表达所必需的.     

转基因(SAG12-IPT)青菜的迟衰特性

利用根癌农杆菌感染方法将融合基因SAG12 IPT导入青菜 ,转基因植株明显表现出衰老延迟的生理现象。SAG12 IPT的抗衰老作用表现为 :在衰老过程中转基因青菜叶片中叶绿素含量高于未转基因的青菜 ,PCR分析结果表明该融合基因已经转入青菜中。激素检测结果表明转基因青菜叶片中细胞分裂素含量高于未转基因植株 ,说明抗衰老与叶片内细胞分裂素含量提高有关。另外 ,转基因植株不仅表现出活体植株衰老延迟 ,而且长在植株上的与离体的叶片滞绿时间延长。这些为蔬菜的耐储存育种提供了新的思路 ,同时为该融合基因在十字花科经济作物中的应用提供了理论依据。     

Existence of S-Glycoprotein-Like Proteins in Anthers of Self-Incompatible Species of Brassica

Experimental evidence is presented that there exists an antherprotein that is reactive with a polyclonal antiserum raisedagainst the stigma S-glycoprotein of the S⁸-homozygote of Brassicacampestris. The antiserum did not react with extracts of seeds,ovaries or leaves. Since this antiserum did not react with thepolysaccharide residues similar to those in S-glycoprotein,it was considered capable of identifying S-glycoprotein-likeproteins in anthers (SA-protein: S-glycopro-tein-like antherprotein). The SA-protein generated a single distinct band ata pI of about 5.0 on blots of gels after isoelectric focusingand three spots at 29 kDa and 83 kDa on blots of two-dimensionalgels, which were different from those of stigma S-glycoprotein.The SA-protein did not contain polysaccharide residue that reactedwith Con A. No allelic differences in pI were found for theSA-protein within a given species, while such differences arecommon in stigma S-glycoprotein. The SA-protein appeared inanthers at the uninucleate microspore stage which is much earlierthan the stage at which the stigma S-xglycoprotein appears.It is present in anther walls rather than in the pollen of matureanthers. The SA-protein is considered to play an important rolein sporophytic control of self-incompatibility. (Received April 2, 1991; Accepted July 24, 1991)     

反义trxs基因导入对小麦籽粒脱支酶活性的影响

以转反义硫氧还蛋白基因株系01TY34-73-9及其对照品种‘豫麦34’为材料,运用PCR检测和酶活性测定的方法,对转基因株系遗传稳定性以及转基因与对照种子中脱支酶活性进行测定。结果显示:(1)外源基因已经稳定遗传至后代;(2)转基因种子在不同成熟时期和不同萌发过程中的脱支酶活性与对照相比均有不同程度的降低平均降低10.3%,但仅花后25 d到收获后5 d脱支酶活性显著低于对照,其中最低值出现在花后30 d,平均比对照下降了12.0%;(3)在花后30 d和后熟5 d萌发过程中,转基因种子脱支酶活性始终低于对照,平均下降6.2%和22.2%。表明反义trxs基因的导入干扰了小麦trxh基因的表达,使trxh转录量减少,小麦籽粒中脱支酶的活性受抑。     

拟南芥冷诱导型启动子CBF 3的克隆及活性检测

目的：构建冷诱导型启动子CBF3基因的植物表达载体,并将其转入烟草。方法：以拟南芥基因组DNA为模板,通过特异PCR扩增,克隆冷诱导表达启动子CBF3（C-repeat binding factor）。用CBF3启动子替换pBI121载体上的35S启动子构建新的载体pBC-GUS,通过农杆菌介导的叶盘法转化烟草。结果：获得了转基因烟草,转基因烟草的GUS组织化学染色及PCR分析结果表明,在低温诱导下,CBF3启动子可增强GUS基因表达。结论：CBF3启动子可应用于植物抗冷基因工程研究。